Inhibitors of folic acid-dependent enzymes

ABSTRACT

The invention relates to compounds of the formula I, or pharmaceutically acceptable salts thereof: 
     
       
         
         
             
             
         
       
         
         
           
             wherein: 
             Z=O or S; 
             n=1-3; 
             R 3 =—CO 2 R 8 , —C(O)SR 8 , —C(O)NHR 8 , —C(S)OR 8 , —C(S)SR 8 , —C(S)NHR 8 , —C(NH)SR 8  or —C(NH)NHR 8 ,
           wherein R 8  is —H or alkyl;   
         
             R 4 =—H, —CH 2 R 5  or —CH 2 CH 2 R 5 ,
           wherein R 5  independently has one of the meanings of R 3 ;   
         
             B=—NR 2 —, —CH 2 NR 2 —, —CH 2 CH 2 NR 2 —, —CH 2 CHR 7 — or —CH 2 O—,
           wherein R 2  is H or a C 1-3  alkyl, alkenyl or alkynyl group, and
               R 7  is H or a C 1-3  alkyl or alkoxy group;   
               
         
           
         
       
    
                         
wherein R 1 =—NH 2  or —OH, and C and D are each, independently, a 5- or 6-membered, substituted or unsubstituted, aromatic or non-aromatic ring which may also contain one or more heteroatoms, and C is connected to group B in any available position.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/663,567, filed Mar. 23, 2007, which claims priority to InternationalPatent Application No. PCT/EP2006/065380, filed Aug. 16, 2006, whichclaims priority to European Application Serial No. EP 05107582.8, filedAug. 17, 2005. Each of the above is incorporated herein by reference.

The present invention relates to a novel class of compounds. Inparticular, the present invention relates to novel compounds thatinhibit enzymes whose natural substrates are folic acid or folic acidderivatives (folates), and that may be used in the treatment of diseasessuch as cancer.

Cancer cells replicate more rapidly than most other cells and so have agreater demand for nucleotides, the precursors of deoxyribonucleic acid(DNA) and ribonucleic acid (RNA). Since all cells do not maintain aresidual store of nucleotides (except for adenosine triphosphate, ATP),they must be synthesized continually during DNA and RNA synthesis.Accordingly, the replication of cancer cells tends to be more sensitivethan that of healthy cells to inhibition of nucleotide biosynthesis, andfor this reason interest is increasing in chemotherapeutic agentscapable of effecting such inhibition.

Nucleotides may be synthesised biologically via de novo pathways fromfundamental metabolic precursors, or via salvage pathways whereby theproducts of nucleic acid degradation, free bases and nucleosides, arerecycled. The de novo pathways are of primary interest with regard tothe search for new chemotherapeutic agents.

The nucleotides 2′-deoxyadenosine-5′-monophosphate (DAMP) and2′-deoxyguanosine-5′-monophosphate (dGMP) are both derived de novo frominosine monophosphate (IMP), which in turn is derived from5-phosphoribosyl-1-pyrophosphate (PPRP). Two enzymes involved in thebiosynthetic pathway between PPRP and IMP are GAR transformylase andAICAR transformylase. GAR transformylase converts glycinamideribonucleotide (GAR) to formylglycinamide ribonucleotide (FGAR) usingN¹⁰-formyltetrahydrofolate, whereas AICAR transformylase uses the samecompound to convert 5-aminoimidazole-4-carboxamide ribonucleotide(AICAR) to N-formylaminoimidazole-4-carboxamide ribonucleotide (FAICAR),as shown below.

The nucleotide 2′-deoxythymidine-5′-monophosphate (dTMP), on the otherhand, is produced by de novo synthesis from2′-deoxyuridine-5′-monophosphate (dUMP), a conversion catalysed by theenzyme thymidylate synthase. During the conversion,N⁵,N¹⁰-methylene-tetrahydrofolate is reduced to 7,8-dihydrofolate; theformer is regenerated via tetrahydrofolate using the enzymesdihydrofolate reductase (DHFR) and serine hydroxymethyl-transferase.These processes are illustrated below.

Interference with these mechanisms has been exploited in the treatmentof cancer. For example, U.S. Pat. No. 2,512,572 discloses a number ofsubstituted pteridines including the potent chemotherapeutic agentmethotrexate, which belongs to the class of “folate antagonists”, andinhibits DNA synthesis by competitively antagonising dihydrofolatereductase, binding with about 100 times higher affinity than its naturalsubstrate, thereby preventing the regeneration of tetrahydrofolate whichis essential for the synthesis of dTMP. This leads to so-called“thymine-less death” in cancer cells. Methotrexate also inhibits GARtransformylase, AICAR transformylase and thymidylate synthase, albeit toa lesser degree. The structures of methotrexate and other relatedanti-folates are shown below.

Compound Structure Methotrexate

Aminopterin

Pemetrexed

Lometrexol

U.S. Pat. No. 4,684,653 discloses compounds of the formula:

wherein R¹ is OH or NH₂, and R³ is H, Me or Et, and their corresponding5,6,7,8-tetrahydro derivatives. These compounds are disclosed to have aneffect on one or more enzymes that utilize folic acid and its metabolicderivatives as a substrate.

U.S. Pat. No. 5,344,932 discloses glutamic acid derivatives of theformula:

wherein R⁵ is H or NH₂, R⁴ is H or OMe, and R² is H or apharmaceutically acceptable cation, and discloses that they have aninhibitory effect on one or more enzymes which utilise folic acid andits metabolic derivatives as a substrate.

U.S. Pat. No. 4,077,957 discloses a method of synthesising variouspteridine compounds, including:

Although such compounds have proved useful in developing new therapeuticstrategies for treating cancer, there are still a number of problemsassociated with their use, including low efficacy, intrinsic andacquired resistance to such drugs in some patients, toxicity and adverseside effects. Consequently, there remains a need for alternativecompounds that can be used in treating cancer and may address one ormore of the aforementioned problems.

Accordingly, in a first aspect of the invention, there is provided acompound of the formula I, or a pharmaceutically acceptable saltthereof:

wherein:

Z=O or S;

n=1-3;

R³=—CO₂R⁸, —C(O)SR⁸, —C(O)NHR⁸, —C(S)OR⁸, —C(S)SR⁸, —C(S)NHR⁸, —C(NH)SR⁸or —C(NH)NHR⁸,

-   -   wherein R⁸ is —H or alkyl;

R⁴=—H, —CH₂R⁵ or —CH₂CH₂R⁵,

-   -   wherein R⁵ independently has one of the meanings of R³;

B=—NR²—, —CH₂NR²—, —CH₂CH₂NR²—, —CH₂CHR⁷— or —CH₂O—,

-   -   wherein R² is H or a C₁₋₃ alkyl, alkenyl or alkynyl group, and        -   R⁷ is H or a C₁₋₃ alkyl or alkoxy group;

-   -   -   wherein R¹=—NH₂ or —OH,            -   C and D are each, independently, a 5- or 6-membered,                substituted or unsubstituted, aromatic or non-aromatic                ring which may also contain one or more heteroatoms, and                C is connected to group B in any available position.

In a second aspect of the invention, there is provided a compoundaccording to the invention in its first aspect, for use in therapy.

In a third aspect of the invention, there is provided a pharmaceuticalcomposition comprising a compound according to the invention in itsfirst or second aspects.

In a fourth aspect of the invention, there is provided the use of acompound according to the invention in its first or second aspects forthe manufacture of a medicament for use in the treatment of a conditionresponsive to inhibition of an enzyme dependent upon folic acid or afolic acid derivative.

In a fifth aspect of the invention, there is provided the use of acompound according to the invention in its first or second aspects forthe manufacture of a medicament for use in the treatment of cancer.

In a sixth aspect of the invention, there is provided a method ofpreparing a compound according to the invention in its first or secondaspects, which comprises the step of:

(a) reacting a compound of the formula IIA-(CH₂)_(m)—X  IIwherein A is as previously defined, m is 0, 1 or 2 and X is a leavinggroup, with a compound of the formula III

wherein Z, n, R², R³ and R⁴ are as previously defined;(b) reacting a compound of the formula IVA-CH₂—X  IVwherein A is as previously defined and X is a leaving group, with acompound of the formula V

wherein Z, n, R³ and R⁴ are as previously defined; or(c) converting one of the following compounds VI or VII into acorresponding organometallic reagent

wherein A, Z, n, R³, R⁴, and R⁷ are as previously defined, and Y is, ineach case independently, a halide, and reacting said reagent with theother of compounds VI or VII.

In a seventh aspect of the invention, there is provided a method ofpreparing a compound according to the invention in its first or secondaspects having the formula I-A or a pharmaceutically acceptable saltthereof

wherein n, R¹, R³ and R⁴ are as previously defined, which comprisesreacting a compound of the formula II-A

wherein X is Cl, Br or I, with a compound of the formula III-A

In an eighth aspect of the invention, there is provided a compound ofthe formula III, V or VII as previously defined, except that R⁴ is—CH₂R⁵ or —CH₂CH₂R⁵.

Preferred embodiments of the invention in any of its various aspects areas described below or as defined in the sub-claims.

In all of the various aspects of the invention, the carbon marked C* maybe asymmetric (when R⁴ is not H) and in this event it will beappreciated that compounds of the formula I may exist in racemic form,or may be separated into their (+) or (−) enantiomers by conventionalmethods. In addition, other chiral centres may be present in somecompounds giving rise to one or more further pairs of enantiomers. Forexample, a second chiral centre exists in those compounds whereinB=—CH₂CHR⁷— wherein R⁷ is a C₁₋₃ alkyl or alkoxy group. All such racemicor enantiomeric forms are intended to lie within the scope of thepresent invention. Furthermore, it will be understood that the compoundsof formula I may exist in one or more tautomeric forms, and each ofthese forms are also intended to lie within the scope of the presentinvention.

As described in more detail hereinafter, the compounds of formula I arestructural analogs of folic acid and have been found to possess activityas inhibitors of those enzymes that are dependent upon folic acid orfolic acid derivatives (folates), such as dihydrofolate reductase(DHFR), at levels comparable to that of methotrexate in vitro. Thecompounds of formula I have also been shown to be active in inhibitingtumor growth in animal models in vivo. It is expected that the latteractivity may be due to the compounds' ability to act as competitiveantagonists of DHFR, although details of the mechanism are not presentedhere. The compounds of formula I may be used in treating cancer, as wellas conditions that are responsive to inhibition of an enzyme dependentupon folic acid or a folic acid derivative.

In preferred compounds of the formula I, one or more of the followingconditions are satisfied:

-   -   Z is O;    -   n is 1;    -   R³ is —CO₂R⁸ and R⁴ is —CH₂CH₂CO₂R⁸;    -   R⁸ is —H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl        or tertiary butyl, preferably —H, -Me or -Et, preferably —H;    -   B is —CH₂NR²—, —CH₂CHR⁷— or —CH₂O—, preferably —CH₂NR²—;    -   R² is —H, -Me, -Et or —CH₂—C≡CH, preferably H;    -   R⁷ is —H, -Me, -Et or —OMe, preferably H.

In addition, preferred compounds of the formula III, V or VII displayone or more of the preferred designations of Z, n, R², R³, R⁴ and/or R⁷set out above.

In group A in formulae I, II, IV or VI, D is preferably a 5-memberedheteroaromatic ring. Preferably, A is

In group A, C may be one of the following groups (points of attachmentto the adjacent ring and to group B are shown):

wherein X is CH or N, and either: Y is C and R⁶ is H, Me, Et or HCO; orY is N and R⁶ is a lone pair of electrons. In preferred embodiments, Xand Y are both N and R⁶ is a lone pair of electrons.

Especially preferred A groups are those of the following structures,which closely mimic those found in naturally occurring pteridines andother heterocyclic bases:

Of particular interest are the following two A groups:

Especially preferred are compounds of the formula I having the above twoA groups, wherein B is —CH₂NR²—, R² is —H, -Me, -Et or —CH₂C≡CH, Z is O,n is 1, and R³ is —CO₂R⁸, preferably any hydrolysable ester group.Individual examples of this group of compounds are set out below.

The compounds of the formula I can be prepared by the methods of theinvention from readily available and inexpensive starting materials. Inthe case where B is —NR²—, —CH₂NR²— or —CH₂CH₂NR²—, for example, thecompounds of formula I can be prepared by coupling a compound of theformula II with a compound of the formula III. The leaving group X willgenerally be a halogen such as chlorine, bromine or iodine, especiallybromine or iodine. This reaction is preferably performed in a dipolaraprotic solvent such as dimethyl formamide (DMF) or dimethylacetamide(DMAc). A basic catalyst such as potassium fluoride may be used, whichaffords a higher yield than tertiary amines or sodium bicarbonate. Wherenecessary, sensitive groups may be protected prior to the reaction usingsuitable protecting groups known in the art, and later deprotected viastandard methods. For example, when R³ is H and R⁴ is CH₂CH₂CO₂H, theseacid groups may be protected for instance as methyl ester groups, withsubsequent deprotection by known methods such as alkaline hydrolysiswith sodium hydroxide in ethanol and precipitation by addition of acid,such as glacial acetic acid. Accordingly, it will be understood that themethod of the invention encompasses the reaction of compounds of theformulae II and III wherein either or both of these compounds are in aprotected form.

In the case where B is —CH₂O—, the compounds of formula I may beprepared, for example, by coupling a compound of the formula IV with acompound of the formula V by a Williamson ether type reaction. In thisreaction, the compound of formula V is generally converted into itsaroxide ion form prior to reaction with the compound of formula IV,using a base such as NaH, for instance. X may be any suitable leavinggroup, in particular a halide.

In the case where B is —CH₂CHR⁷—, the compound of formula I may beprepared, for example, by coupling a compound of the formula VI with acompound of the formula VII by any known carbon-carbon bond formingreaction, especially those involving the use or formation oforganometallic reagents such as Grignard reagents and lithium orcopper-lithium compounds. For instance, a compound of the formula VIImay be converted into its corresponding Grignard reagent or lithiumcuprate reagent and reacted with a compound of the formula VI.Alternatively, a compound of the formula VI may be converted into itscorresponding Grignard reagent or lithium cuprate reagent and reactedwith a compound of the formula VII. Once again, suitable protectinggroups for any reactive substituent groups will be well-known to thoseskilled in the art.

The intermediates II to VII may be prepared by conventional methods. Byway of illustration, compounds of the formula III, V or VII may beprepared by reacting a compound of the formula

with a compound of the formula

wherein B′ is —NHR², —OH or —CHYR⁷ and X is a leaving group, in thepresence of a base. This may be followed by removal of the cyano groupby hydrolysis and decarboxylation, with the use of suitable protectinggroups where necessary.

Compounds of formula I have an inhibitory effect on one or more enzymeswhich utilize folic acid, and in particular metabolic derivatives offolic acid as a substrate. These enzymes include GAR transformylase,AICAR transformylase, dihydrofolate reductase and thymidylate synthase.The compounds appear to be particularly active as inhibitors ofdihydrofolate reductase. They can be used, alone or in combination, totreat neoplasms which in the past have been treated with methotrexate,including choriocarinoma, leukaemia, adenocarcinoma of the femalebreast, epidermid cancers of the head and neck, squamous or small-celllung cancer, and various lymphosarcomas. Although not wishing to belimited by theory, it is believed that the modified ketomethylenic orthioketomethylenic side chain of the compounds of formula I leads to alower renal toxicity compared with methotrexate. Inactivation due tohydrolysis is minimal due to the lower lability of the ketomethylenic orthioketomethylenic group, allowing a longer half-life. In addition,compounds of the formula I exhibit improved physico-chemicalcharacteristics compared with those of the prior art.

As discussed above, primary interest in the compounds of the inventionrelates to their use in the treatment of cancer, since cancerous cellsreplicate more rapidly than healthy cells and so have a greater demandfor nucleotides. The therapeutic utility of the compounds of theinvention extends further than this, however, since all fast-growingcells have a similar high demand for nucleotides. For example,methotrexate has been used in the treatment of psoriasis and mycosisfungoides, and in inducing miscarriage in patients with ectopicpregnancy. Methotrexate has also been used in the treatment ofrheumatoid arthritis, although the mechanism of action in this instanceis not fully understood.

The compounds may be administered either orally or preferablyparenterally, alone or in combination with other anti-neoplastic agentsor with other therapeutic agents, such as steroids, to a mammal,preferably a human, suffering from neoplasm and in need of treatment.Parenteral routes of administration include intramuscular, intrathecal,intravenous or intra-arterial.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more fully understood, it will now bedescribed by way of example only with reference to the accompanyingdrawing, wherein

FIG. 1 is a schematic representation of the total synthesis of twocompounds according to the present invention (compounds 3 and 4), asdescribed in detail below in Examples 1 and 2.

The following examples are intended to demonstrate the invention but arenot intended to limit the invention in any manner.

EXAMPLE 1 Synthesis of Compound 3

Source of Starting Materials

3-chloropropanoyl chloride and ethyl cyanoacetate were obtainedcommercially from Sigma-Aldrich Company Ltd., The Old Brickyard, NewRoad, Gillingham, Dorset SP8 4XT, United Kingdom, or synthesised bystandard methods. α-bromo-p-nitro-acetophenone 7 was obtained bybromination of p-nitroacetophenone with bromine in tetrahydrofuran(THF). 2,4-diamino-6-bromomethylpteridine 2 was obtained by standardmethods (see, for example, U.S. Pat. No. 4,077,957 and U.S. Pat. No.4,224,446).

Step A Synthesis of Compound 6

3-chloropropanoyl chloride was esterified with ethanol in the presenceof pyridine or triethylacetate to produce ethyl 3-chloropropionate 5.The latter was condensed with ethyl cyanoacetate according to theprocedure of L. Ruzicka et. al., Helv. Chim. Acta 17, 183-200 (1934), CA28:2584, or Koelsch, C. F., J. Am. Chem. 65, 2458-9 (1943), to formdiethyl α-cyanoglutarate 6. ¹H-NMR confirmed the expected structure. GC:97% purity.

Step B Synthesis of Compound 8

175 g (0.71 mmol) α-bromo-p-nitro-acetophenone 7 was added in portionsat 0-5° C. to a suspension of 175 g (0.82 mmol) diethyl α-cyanoglutarate6 and 175 g (3 mmol) KF in 500 ml DMF. The reaction was monitored bythin layer chromatography (TLC). After 4 hours, the reaction mixture wassuspended in 2 l of water containing 0.1% acetic acid at pH 5. Afterdecanting the water, the gummy precipitate was washed with water (2×750ml) then triturated with 300 ml methanol. When crystallisation wascomplete, the precipitate was filtered and washed successively with anexcess of methanol and ether, affording 210 g of compound 8, a yellowsolid with m.p. 92.1° C. (Yield 68%) After chromatography on silica gel(50:50:5 benzene-cyclohexane-ethanol) the product had m.p. 99.7° C. TLCon silica gel plates (5:1:3:10:0.1 benzene-ethanol-cyclohexane-petroleumether-AcOH) showed a single spot with Rf (retention factor) 0.38. HPLC:97% purity.

Step C Synthesis of Compound 9

30 g (0.08 mmol) compound 8 was dissolved in 400 ml methanol andhydrogenated in a hydrogenation flask at room temperature in thepresence of 6 g 20% Pd/C catalyst. The theoretical volume of hydrogen(c. 6200 ml; 0.28 mmol) was absorbed in 1 hour (TLC control). Theplatinum catalyst was filtered and the methanol was evaporated. Thecrude product obtained solidified on drying in vacuo, resulting in 27.6g compound 9, a yellow solid (yield 99%) which was used without furtherpurification in the conversion to crude compound 10 described below. Thepurity was acceptable by TLC analysis. TLC (4:1 chloroform-methanol)showed a single spot, Rf 0.5 (characteristic reaction with4-dimethylamino benzaldehyde). The HCl salt was isolated after reflux inHCl. LC-MS and ¹-H NMR confirmed the expected structure; HPLC: 99%purity.

Step D Synthesis of Compound 10

A solution of 52.2 g (0.15 mmol) intermediate 9 in 1000 ml methanol wasprepared. 188 ml 6N NaOH was added dropwise at room temperature for 1hour and the solution was allowed to stand for 12 hours. The reactionmixture was then diluted with 300 ml water and concentrated under highvacuum. 700 ml 37% HCl was added to the residue and the resultingmixture was heated to reflux for 4 hours. The mixture obtained wasdiluted with 1.5 l methanol and the NaCl precipitate was removed byfiltration. The filtrate was used in step E. A small amount of diacid 10was isolated before dilution, by filtering the suspension and washingthe precipitate successively with an excess of water, acetone and ether.TLC (4:1 chloroform-methanol) showed a single spot, Rf 0.26.

Step E Synthesis of Compound 1

The methanolic solution of the dicarboxylic acid 10 obtained in step Dwas cooled at 0-5° C. and 100 ml thionyl chloride was added dropwise.The reaction mixture was stirred under reflux for 3 hours, then cooledto room temperature and the solvent was evaporated off. The precipitateobtained was filtered and washed with ether, resulting in 27 g compound1 (yield 63%), a solid with m.p. 115-116° C. After recrystallisationfrom tetrahydrofuran, 17.5 g white crystals of 1 were obtained havingm.p. 116-117° C. TLC (4:1 chloroform-methanol) showed a single spot, Rf0.73. UV spectra: 234, 319 nm (MeOH). ¹H NMR spectra: 2.0 (2H, m, CH₂CH₂COOCH₃), 2.5 (2H, t, CH₂CH ₂COOCH₃), 3.1 (2H, m, COCH ₂), 3.5 (1H,m, COCH₂CH), 3.75 (6H, s, COOCH ₃), 7.6-8.0 (4H, m, CH arom.). HPLC: 99%purity.

Step F Synthesis ofN-[4-[[2,4-diamino-6-pteridinyl)methyl]amino]benzoyl] pseudoglutamicester (Compound 3)

A mixture of 7 g (27.4 mmol) 2,4-diamino-6-bromomethylpteridine 2 and 7g (23.4 mmol) dimethyl N-[4-methyl-amino)benzoyl]pseudogluamate 1 in 70ml N,N-dimethylacetamide was stirred for 30 minutes at 70° C. thenallowed to stand at room temperature overnight protected from light,then heated again for 10 minutes to 100° C. The reaction was controlledby TLC. After cooling, the reaction mixture was poured into wateracidified with AcOH at pH 4 (1000 ml). The dark-yellow precipitate thatformed was filtered and washed three times with water and allowed to airdry. 2.6 g orange-yellow product 3 was obtained, m.p. 200-210° C. Thefiltrate was treated with 10% NaHCO₃ and the precipitate formed wasseparated in the same manner, resulting in a second fraction of dimethylester 3 (2 g). Total yield: 36%. TLC (4:1 chloroform-methanol) showed asingle spot, Rf 0.48. UV Spectra: 210, 242, 332 (0.1 N HCl); 238, 335(MeOH).

EXAMPLE 2 Synthesis ofN-[4-[[2,4-diamino-6-pteridinyl)methyl]amino]benzoyl] pseudoglutamicacid (Compound 4)

1 g (2.1 mmol) dimethyl ester 3 was added in portions to a solution of10 ml 2N NaOH and 25 ml ethanol, and the mixture was stirred at roomtemperature for 4 hours. The precipitate formed was filtered anddissolved in distilled water. The alkaline solution was treated withcharcoal, filtered and the pH was adjusted to 4.5 with 10% AcOH. Theprecipitate was filtered and washed with water at pH 4.5, then withacetone, resulting in 0.8 g compound 4 (yield 85%). The product, a brownsolid, was purified by preparative HPTLC (High Performance Thin LayerChromatography). After elution with 50:50:5 CH₃CN—H₂O—NH₄OH, the diacidwas extracted from silica gel with 100 ml NaOH solution at pH 8. Thewater was removed by freeze-drying. TLC (7:2:1 CH₃CN—H₂O—NH₄OH) showed asingle spot, Rf 0.80. Mass spectrum: m/z 120 (M+, 100%). IR spectra(KBr): 1651 (COCH), 1594 (C═C), 1563, 1403 (C═O acid), 1176 (C—O), 823(CH). UV spectra: 242, 332 nm (0.1 N HCl); 232, 259, 325 nm (0.1 NNaOH); 229, 262, 318 (MeOH). ¹H NMR: 1.6 (3H, m, CH—CH ₂), 2.2 (2H, t,CH₂CH ₂COOH), 2.9 (2H, m, COCH ₂), 4.6 (2H, s, CH ₂NH), 6.8-7.8 (4H, m,CH arom.), 9 (1H, s, 7-CH). HPLC: 97% purity.

Other compounds of formula I can be produced by adapting the proceduresset out above in an appropriate manner. For example, intermediate 1 andanalogous compounds can be converted to their corresponding N-methylderivatives by reaction with formaldehyde and sodium cyanoborohydride.Intermediate 6 can also be produced by reacting ethyl cyanoacetate withethyl acrylate according to standard procedures.

EXAMPLE 3 In Vitro Inhibition of DHFR

The ability of compounds of formula I to inhibit dihydrofolate reductase(DHFR) in vitro was measured using a standard DHFR enzyme inhibitionassay. DHFR enzyme was purified from rat livers or the commerciallyavailable DHFR was used, this being produced by recombinant expressionin E. coli. Assays of enzyme activity were performed at 37° C. bymonitoring changes in UV absorbance at 340 nm of a solution containing50 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 7.0), 1mM EDTA, 75 μM 2-mercaptoethanol, 0.1% bovine serum albumin, 20 μMdihydrofolate, and 100 μM NADPH. Reactions were initiated by addingdihydrofolate. Each titration of the inhibitor was performed twice, andmean DHFR activity was plotted against inhibitor concentration to obtainIC₅₀ values. The ratio of IC_(50(Compound))/IC_(50(MTX)) was designatedrelative IC₅₀ and the results of one representative experiment (out of 5experiments) for compounds 438 and 497 relative to methotrexate (MTX)are shown in Table 1. The results obtained overall suggest that mostcompounds of formula I tested possessed an in vitro potency similar tothat of MTX.

TABLE 1 Compound Chemical formula Relative IC₅₀ MTX 1 438 Compound 4, R³= H 12.5 497 Compound 4 0.75

N.B. Compound 438 has the same structure as compound 4 in FIG. 1 exceptthat R³ (—CH₂CH₂CO₂H) is replaced by H.

EXAMPLE 4 In Vitro Cytotoxicity

The cytotoxicity of compounds of formula I against a number of tumorcell lines (CCRF-CEM, HepG2, HeLa, KB, L1210, A549 and COLO205) wasassayed by measuring cell viability at different time points followingdrug addition up to 72 hours, and compared with the correspondingcytotoxicity of methotrexate and pemetrexed (obtained as Alimta®). Thecompounds of formula I demonstrated potent inhibitory effects againstgrowth of all the cell lines tested, with the strongest inhibitoryeffects being seen against the L1210 cell line. Compared withmethotrexate and pemetrexed, the compounds of formula I exhibitedsimilar or stronger inhibitory effects against all the cell lines, andin some cases the onset of cytotoxicity was more rapid. Further,compounds of formula I wherein B=—CH₂NH— were found to be particularlycytotoxic.

Cytotoxicity was not reversed by addition of purines, such ashypoxanthine, or by addition of aminoimidazole carboxamide (up to veryhigh concentration), but was reversed by addition of leucovorin,indicating cytotoxicity was due to antagonism of a folate-relatedmechanism. Consistent with a proposed mechanism of action in which DHFRis the main target, the addition of thymidine reversed cytotoxicityinduced by the compounds of formula I only at high concentrations. Theseeffects indicate specific inhibition of de novo purine synthesis and aless significant inhibition of the thymidylate cycle, however morepronounced than in the case of methotrexate. The compounds of formula Ialso inhibited glycinamide ribonucleotide transformylase in a comparableconcentration range to methotrexate.

EXAMPLE 5 Tumor Inhibition in Animal Models In Vivo

The ability of compounds of formula I to inhibit tumor growth in micewas tested as follows. 5×10⁶ L 1210 cells were injected subcutaneouslyin the axillary region of DBA/2 mice (groups of 8 mice/treatment).Following intraperitoneal administration of saline solution only orsaline solution containing a compound of formula I the length and widthof the control tumor (receiving only saline) was measured at theindicated time and compared to those of animals receiving test compoundto calculate the percentage of inhibition. Intraperitonealadministration of a saline solution containing the test compound, dailyfor 6 days, led to 60% tumor-free long term survivors (tumor weightzero). Median survival times for saline-treated control animals andanimals receiving Compound 4 (0.5 mg/kg) were 6.7 and 15.6 days,respectively. Oral administration of the compound required a higherdosage of the inhibitor and led to a less-marked, but still significantreduction of the tumor weight and 25% long-term survivors as comparedwith the saline-treated control group.

Compounds of formula I were also active in vivo against the carcinomaW256 (TGI=28%), most likely due to the higher solubility and thuspassive transport into the tumor compared with other anti-folate agents.Median survival times for saline-treated control animals and animalsreceiving Compound 4 (1 mg/kg) intraperitoneally in the Walker-256 rattumor model were 22.5 and >46.3 days, respectively.

The tumor evolution was measured following different treatment regimesand results are summarized in Table 2.

TABLE 2 Effect of various concentration of Compound 4 in a rat tumormodel No. animals surviving at day 30 days post- Tumor volume GroupTreatment transplant (cm³) ± SE 1 Saline Control 2/10 37.3 ± 0.3 2 0.5mg/kg - 3 doses 9/10  13.4 ± 3.07 every 4 days 3 0.25 mg/kg - 3 doses8/10   23 ± 3.5 every 4 days 4 0.1 mg/kg - 5 doses 10/10  14.5 ± 3.8every day 5 1 mg/kg - one dose 8/10 17.1 ± 3.3

1. A method of preparing a compound, which comprises the step of:reacting a compound of the formula IIA-(CH₂)_(m)—X  II wherein A is

wherein R¹=—NH₂ or —OH, C and D are each, independently, a 5- or6-membered, substituted or unsubstituted, aromatic or non-aromatic ringwhich may also contain one or more heteroatoms, m is 0, 1 or 2 and X isa leaving group, with a compound of the formula III

wherein Z is O or S; n is 1-3; R² is H or a C₁₋₃ alkyl, alkenyl oralkynyl group; R³ is —CO₂R⁸, —C(O)SR⁸, —C(O)NHR⁸, —C(S)OR⁸, —C(S)SR⁸,—C(S)NHR⁸, —C(NH)SR⁸ or —C(NH)NHR⁸, wherein R⁸ is —H or alkyl; and R⁴ is—H, —CH₂R⁵ or —CH₂CH₂R⁵, wherein R⁵ independently has one of themeanings of R³, to produce a compound of formula I:

wherein: Z=O or S; n=1-3; R³=—CO₂R⁸, —C(O)SR⁸, —C(O)NHR⁸, —C(S)OR⁸,—C(S)SR⁸, —C(S)NHR⁸, —C(NH)SR⁸ or —C(NH)NHR⁸, wherein R⁸ is —H or alkyl;R⁴=—H, —CH₂R⁵ or —CH₂CH₂R⁵, wherein R⁵ independently has one of themeanings of R³; B=—NR²—, —CH₂NR²—, —CH₂CHR⁷— or —CH₂O—, wherein R² is Hor a C₁₋₃ alkyl, alkenyl or alkynyl group, and R⁷ is H or a C₁₋₃ alkylor alkoxy group; A=

wherein R¹=—NH₂ or —OH, C and D are each, independently, a 5- or6-membered, substituted or unsubstituted, aromatic or non-aromatic ringwhich may also contain one or more heteroatoms, and C is connected togroup B in any available position.
 2. A method as claimed in claim 1,wherein Z is O.
 3. A method as claimed in claim 1, wherein n is
 1. 4. Amethod as claimed in claim 1, wherein R⁸ is —H, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl or tertiary butyl.
 5. A method as claimedin claim 1, wherein R³ is —CO₂R⁸ and R⁴ is —CH₂CH₂CO₂R⁸.
 6. A method asclaimed in claim 5, wherein R⁸ is H, methyl or ethyl.
 7. A method asclaimed in claim 1, wherein R² is —H, -Me, -Et or —CH₂C≡CH.
 8. A methodas claimed in claim 1, wherein D is a 5-membered aromatic ring.
 9. Amethod as claimed in claim 1, wherein A is


10. A method as claimed in claim 1, wherein C is:

wherein X is CH or N, and either: Y is C and R⁶ is H, Me, Et or HCO; orY is N and R⁶ is a lone pair of electrons.
 11. A method as claimed inclaim 10, wherein A is of the formula A-i or A-ii:


12. A method as claimed in claim 11, wherein A is of the formula A-i-1or A-i-1:


13. A method as claimed in claim 12, wherein R² is —H, -Me, -Et or—CH₂C≡CH; Z is O; n is 1; and R³ is —CO₂R⁸.
 14. A method as claimed inclaim 11, wherein R² is —H, -Me, -Et or —CH₂C≡CH; Z is O; n is 1; R³ is—CO₂R⁸; R⁴ is —H or —CH₂CH₂CO₂R⁸; R⁸ is independently —H, -Me or -Et; ifA is A-i and Y is C, R⁶ is —H; and if A is A-ii, R¹ is —OH.